In the field of genome science, the emphasis has recently shifted from “structural analysis” involving the identification of gene sequences to “functional analysis” using gene expression products. This is because proteins and other gene products are basically provided with gene functions. For this reason, protein analysis is essential for analyzing the functions of genes. Analysis of protein function is carried through, for example, biochemical functional analyses by analyzing protein-protein interactions, protein-nucleic acid interactions and so on.
Known examples of methods for analyzing protein-protein interactions include the yeast two-hybrid method (Chien, C. T. et al., Proc. Natl. Acad. Sci. USA, 88, 9578-9582 (1991)), the phage display method (Smith, G. P., Science, 228, pp. 1315-1317 (1985)), the GST-fusion protein pull-down method, and the immunoprecipitation method or the like. Known examples of methods for analyzing protein-nucleic acid interactions include electrophoretic mobility shift assays (Revzin, A., et al., Anal. Biochem., 153, 172 (1986)), the DNase I footprint method (Calas, D., et al., Nucleic Acid Res., 5, 3157 (1978)) and the methylation buffer method.
In addition, methods for analyzing protein interactions are also being developed using an in vitro virus method (see Nemoto, et al., FEBS Lett., 414, 405 (1997); Tabuchi, et al., FEBS Lett., 508, 309 (2001)) utilizing specific properties of puromycin (see WO 01/016600).
The in vitro virus (IVV) method is a powerful protein evolutionary engineering technique involving the selection of a peptide that specifically bonds to a specific molecule (not limited to proteins) in a library of a vast number of peptides or proteins having random sequences. However, the high cost and inefficiency of “linkers” for ligating puromycin to mRNA is a major obstacle to practical use of this technique. It is important for practical linkers to satisfy conditions such as (1) good ligation efficiency between the linker and mRNA, (2) being able to be purified immediately from a translation system, having a reverse transcription DNA primer and being able to be rapidly converted to DNA, and (3) easy purification of converted DNA/RNA-protein conjugates. In order to efficiently proceed with the reverse transcription of (2) above in particular, it is important to translate using a biotin-labeled linker followed by promptly purifying the mRNA-protein conjugate from a cell-free translation system.
However, there is as of yet no known linker that simultaneously satisfies these three conditions, thus resulting in the need for the development of such a linker as well as mRNA/cDNA-puromycin-protein conjugates constructed using that linker.